WO2001030993A1 - Procede de detection d'un acide nucleique cible - Google Patents

Procede de detection d'un acide nucleique cible Download PDF

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Publication number
WO2001030993A1
WO2001030993A1 PCT/JP2000/007430 JP0007430W WO0130993A1 WO 2001030993 A1 WO2001030993 A1 WO 2001030993A1 JP 0007430 W JP0007430 W JP 0007430W WO 0130993 A1 WO0130993 A1 WO 0130993A1
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
primer
labeled
target nucleic
extension product
Prior art date
Application number
PCT/JP2000/007430
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English (en)
Japanese (ja)
Inventor
Yasumasa Mitani
Original Assignee
Wakunaga Pharmaceutical Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Wakunaga Pharmaceutical Co., Ltd. filed Critical Wakunaga Pharmaceutical Co., Ltd.
Priority to AU79558/00A priority Critical patent/AU7955800A/en
Publication of WO2001030993A1 publication Critical patent/WO2001030993A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Definitions

  • the present invention relates to a method and a kit for specifically detecting a target nucleic acid in a biological sample.
  • PCR polymerase chain reaction method
  • RT-PCR RT-PCR
  • the gene amplification method represented by the reverse transcriptase polymerase chain reaction method has been widely studied and used as a method with high detection sensitivity.
  • a hybridization method in which a single or double strand having a base sequence complementary to a target nucleic acid is labeled with a radioactive or non-radioactive labeling substance and the target sequence is detected using complementarity with the target sequence is also known. It is widely used as a method for detecting the presence of a target nucleic acid or a target nucleic acid amplification product.
  • a liquid-solid hybridization method TR Gingeras et al., Nucleic Acid Res.
  • amplification of the target DNA or RNA can be performed in a short time, but detection of the amplified DNA fragment is complicated. However, it takes a long time until the hybridization method is performed. In the hybridization method, the operation is complicated and time-consuming, and a satisfactory method in terms of sensitivity and the like has not been obtained.
  • a method of amplifying a nucleic acid using a technique such as PCR using a first labeled nucleic acid primer as a sense primer and a second labeled nucleic acid primer labeled with a label different from the first labeled nucleic acid primer as an antisense primer has a disadvantage that when a non-specific amplification product other than the target nucleic acid amplification product is obtained, it is finally detected as a false positive. Disclosure of the invention
  • An object of the present invention is to provide a method and a kit for detecting a target nucleic acid quickly and with high sensitivity by a simple operation method.
  • the present inventors have conducted intensive studies on a method for detecting a nucleic acid that does not require a complicated hybridization reaction, and as a result, have found that a primer extension product labeled with two types of labels can be obtained. After that, it was found that the target nucleic acid could be detected simply, quickly and with high sensitivity by using affinity chromatography, and the present invention was completed. That is, the present invention relates to a method for specifically detecting a target nucleic acid in a biological material,
  • the present invention also provides a kit for specifically detecting a target nucleic acid in a biological material
  • the method for detecting a target nucleic acid according to the present invention comprises the above steps (a) to (d).
  • a known nucleic acid amplification reaction can be involved.
  • Such a nucleic acid amplification method is not particularly limited, and examples thereof include a ligase chain method (LCR) and a PCR method (Japanese Patent Application Laid-Open No. 62-281), with the PCR method being particularly preferred.
  • the primer set refers to a combination of a sense primer and a primer (antisense primer) complementary to a target nucleic acid sequence on the opposite side, and in the present invention, one or both of them are the first. It only needs to be labeled.
  • the first labeled nucleic acid primer and the second labeled nucleic acid primer used in the method for detecting a target nucleic acid of the present invention need to have different base sequences and have different labels.
  • the second labeled nucleic acid primer recognizes a part of a sequence other than the primer sequence in the extension product extended by the first labeled nucleic acid primer. As a result, only when annealing occurs, a double-stranded primer extension product having both labeled substances (both labeled primer extension products) is formed, and the target nucleic acid is detected.
  • the melting temperatures of the respective extension products will be different. By performing the annealing and extension reactions at the respective optimum annealing temperatures, both ends of the primer dimer and the like will be obtained. The generation of non-specific reaction by-products with different labels can be excluded.
  • the label must be one that specifically binds to the specific binding reagent present on the chromatography support.
  • Specific binding reagents and combinations of substances that specifically bind to it Examples of the combination include a combination of biotin and streptavidin or avidin, a hapten and an antibody, a ligand and a receptor, and the like. Among them, it is generally preferable to use an oligonucleotide having high heat stability and a small molecular size as the oligonucleotide.
  • the primer in the case of biotin and streptavidin, it is preferable to label the primer with biotin and apply the streptavidin to the chromatographic carrier, and the primer is attached to the chromatographic carrier by binding of the biotin and streptavidin. Join.
  • the label of either the first labeled nucleic acid primer or the second labeled nucleic acid primer must be capable of binding to a specific binding reagent immobilized at a predetermined position (detection region) of the chromatography carrier.
  • examples of such a label include DNP against an immobilized anti-DNP antibody.
  • nucleic acid used for the labeled nucleic acid primer a natural nucleic acid may be used as it is, or a synthetic oligonucleotide sequence may be used.
  • the position of the label in the primer may be any site that does not significantly affect the efficiency of the extension reaction of the primer, and preferably includes an active group in a hydroxyl group, a base, or a phosphodiester moiety near the 5 'end.
  • first labeled nucleic acid primer and the second labeled nucleic acid primer one labeled with a specific binding substance for detection such as biotin as the first labeled nucleic acid primer, and immobilized with DNP or the like as the second labeled nucleic acid primer
  • a primer labeled with a substance capable of binding to a reagent and having a sequence length different from that of the first labeled nucleic acid primer by 3 to 15 mer is selected.
  • Annealing by contacting the target nucleic acid with the first labeled nucleic acid primer and The extension reaction (step (a)) can be carried out under the conditions used for ordinary annealing.
  • the extension reaction is carried out at 30 to 80 ° C for 5 seconds, preferably at 35 to 72 for 10 to 60 seconds. You.
  • the primer extension product annealed and extended by the first labeling nucleic acid primer, and the nucleic acid amplification product amplified by the amplification reaction are complemented with ⁇ type.
  • This is performed using a typical second-labeled nucleic acid primer.
  • the target nucleic acid and the first labeled nucleic acid primer extension product in step (a) are used. It must be performed at a temperature lower than the melting temperature of
  • a double-stranded primer extension product having both labeled substances can be obtained, and the double-stranded nucleic acid can be used for the labeling of the first and second labeled nucleic acid primers.
  • a chromatographic carrier having a specific binding reagent such as an antibody that specifically binds
  • the presence / absence of the target nucleic acid can be detected simply, quickly and specifically. That is, in the method for detecting a target nucleic acid of the present invention, the target nucleic acid detected on the chromatography carrier is only a double-stranded nucleic acid having both labeled substances. If a specific amplification product is obtained, it will not be detected.
  • the so-called affinity mouth method is used for detection of both labeled primer extension products.
  • a specific binding reagent such as an antibody that binds to one of the double-stranded nucleic acids having both labels at a predetermined position (detection region) of the chromatography carrier is immobilized, and the other is used.
  • a specific binding reagent that can bind to a nucleic acid labeled product is kept in a dry state so that it can move when wet on the upstream side of the chromatography medium, and the target nucleic acid amplification product that has a labeled product at both ends is different. Is developed from the upstream of the chromatographic carrier by utilizing the capillary phenomenon.
  • the target nucleic acid extension and the specific binding reagent develop first while reacting, and finally are detected.
  • This is a method of capturing and detecting a target nucleic acid in the form of a sandwich-like complex in a region.
  • the target nucleic acid can be detected in an extremely short time as compared with the method of performing hybridization, for example, as short as 10 minutes or less as described in Examples below. .
  • the detection method is simple, and it does not require special techniques or equipment, so that multiple samples can be tested simultaneously. Moreover, the confirmation can be done visually and is extremely simple.
  • the chromatographic carrier used must have a pore size large enough to allow the target amplified nucleic acid to move stably and satisfactorily on the chromatographic carrier, to be sufficiently developed, and to reach the detection region without fail.
  • examples thereof include glass fiber filter paper, cellulose membrane, nitrocellulose membrane, and nylon membrane.
  • the specific binding reagent immobilized on the chromatography carrier is immobilized on a predetermined detection region, and binds to the target nucleic acid label in this region.
  • the specific binding reagent immobilized on the detection region include an antibody against hapten, for example, an anti-DNP antibody against DNP.
  • such an antibody may be either a monoclonal antibody or a polyclonal antibody.
  • the particles of the particulate specific binding reagent include colloid particles, inorganic particles, ceramic particles, metal or oxide particles thereof, gel particles, synthetic polymer particles, dye sol, and the like. From the viewpoint of dispersion stability, metal colloid particles such as gold colloid and synthetic polymer particles such as latex particles are particularly preferable.
  • the specific binding reagent is as described above, and avidin, streptavidin and the like are preferable.
  • the biological material used in the present invention may be any material as long as it contains the nucleic acid of interest. Examples of sources of such material include plants, insects, and animals. , Serum, plasma, cerebrospinal fluid, biopsy, tissue culture cells Examples thereof include a growth medium from tissue culture cells, a tissue extract, and a membrane washing solution.
  • the toxin type 1 gene (VT 1) of enterohemorrhagic Escherichia coli (E.co1i) was selected as the target nucleic acid.
  • a first labeled nucleic acid primer set and a second labeled nucleic acid primer as described below were used.
  • the primer on the sense side of the first labeled nucleic acid primer was a label capable of binding to colloidal gold-labeled streptavidin, and was introduced with a biotin at the 5 ′ end, while the primer on the antisense side was unlabeled.
  • DNP was introduced at the 5 ′ end as a label capable of binding to the immobilized anti-DNP antibody on the affinity-mouthed chromatography medium.
  • Imnodine ABC (Paull) was selected as a chromatographic carrier, and anti-DNP mouse monoclonal antibody was adjusted to 5 mg mL with 10 mM phosphate buffered saline, and dispenser Shotmaster II (Musashi Engineering) Then, it was coated in a line so as to have an I LZI cm, air-dried, gelatin-blocked, washed, and dried under negative pressure. The reaction film and the above-mentioned streptavidin pad labeled with colloidal gold were adhered on a tape to obtain an immunochromatography carrier. The obtained immunochromatographic medium was stored in an aluminum bag together with silica gel.
  • Enterohemorrhagic Escherichia coli with overnight cultured Vero toxin type 1 gene (VT 1) E. co 1 i
  • 1 X 1 0 6 with sterile water 1 X 1 0 5, 1 X 1 0 4, 1 X 1 It was adjusted to 0 3 ce 11 s ZmL.
  • These 100 were collected in 1.5 mL tubes, heated at 95 for 10 minutes, and subjected to nucleic acid amplification using 10 L of the solution as a target nucleic acid sample.
  • the target nucleic acid amplification product was detected by the following method. As a comparison, detection was performed by electrophoresis.
  • the above-mentioned second labeled nucleic acid primer 50 ng ZD, IL was added to the PCR reaction tube. Using a PCR device, heat denaturation was performed at 98 for 3 minutes, annealing at 65 for 30 seconds, and extension reaction at 72 for 30 seconds. Each reaction solution was developed on the immunochromatography carrier described above, and after 5 minutes, the presence or absence of color development was visually determined.
  • the method for detecting a target nucleic acid according to the present invention can greatly reduce the analysis time by performing an extension reaction using primers without performing a complicated operation in a so-called hybridization reaction.
  • the use of affinity chromatography makes it possible to analyze a sample simply, quickly and with high sensitivity.
  • the sample is in a roughly purified state and the sample can be easily prepared.
  • the primers can be completely adjusted to the target nucleic acid by appropriately adjusting the reaction conditions such as polymerase.
  • a distinction can be made between complementary and non-complementary cases. That is, point mutations can be easily detected without using the hybridization method or the like.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de détection d'un acide nucléique cible présent dans un matériau biologique. Ce procédé consiste à : (a) mettre en contact l'acide nucléique cible ou une chaîne complémentaire de celui-ci avec une première amorce d'acide nucléique marquée complémentaire ou avec un premier ensemble contenant cette amorce en vue de former ainsi un produit d'extension de ladite amorce ;(b) dénaturer le produit d'extension de l'amorce d'acide nucléique comme cela est décrit ci-dessus afin d'obtenir une chaîne dénaturée ;(c) mettre en contact cette chaîne dénaturée avec une seconde amorce d'acide nucléique marquée complémentaire de celui-ci afin de former un produit d'extension d'amorce à double-marquage et enfin (d) développer ce produit d'extension d'amorce à double-marquage sur un support chromatographique possédant des réactifs de liaison qui s'associent en particulier aux marques de la première amorce d'acide nucléique marquée ainsi qu'à celles de la seconde amorce d'acide nucléique marquée et un assemblage de détection d'un acide nucléique cible par ce procédé. L'utilisation de ce procédé/assemblage de détection permet de détecter, de manière pratique, rapide et très précise la présence ou l'absence d'acide nucléique cible dans un échantillon.
PCT/JP2000/007430 1999-10-25 2000-10-24 Procede de detection d'un acide nucleique cible WO2001030993A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU79558/00A AU7955800A (en) 1999-10-25 2000-10-24 Method of detecting target nucleic acid

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP30239999 1999-10-25
JP11/302399 1999-10-25

Publications (1)

Publication Number Publication Date
WO2001030993A1 true WO2001030993A1 (fr) 2001-05-03

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009034842A1 (fr) * 2007-09-11 2009-03-19 Kaneka Corporation Procédé de détection d'acide nucléique, et coffret de détection d'acide nucléique
WO2009054510A1 (fr) 2007-10-25 2009-04-30 Riken Procédé d'amplification isotherme et adn polymérase utilisée dans ce procédé
WO2010061922A1 (fr) 2008-11-27 2010-06-03 独立行政法人理化学研究所 Nouvelle protéine muts et procédé d'utilisation de celle-ci pour déterminer des mutations

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0439222A2 (fr) * 1990-01-26 1991-07-31 Johnson & Johnson Clinical Diagnostics, Inc. Réactif insoluble, sonde d'acide nucléique, kit de test et procédés de diagnostic et purification
JPH05176800A (ja) * 1991-12-30 1993-07-20 Bio Sensor Kenkyusho:Kk 成人ｔ細胞白血病ウィルス遺伝子の検出方法
WO1994026934A2 (fr) * 1993-05-06 1994-11-24 Baxter Diagnostics Inc. Methode de detection du papillomavirus chez l'homme
JPH0775599A (ja) * 1993-09-07 1995-03-20 Japan Synthetic Rubber Co Ltd 標的核酸の検出方法およびそのためのキット

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0439222A2 (fr) * 1990-01-26 1991-07-31 Johnson & Johnson Clinical Diagnostics, Inc. Réactif insoluble, sonde d'acide nucléique, kit de test et procédés de diagnostic et purification
JPH05176800A (ja) * 1991-12-30 1993-07-20 Bio Sensor Kenkyusho:Kk 成人ｔ細胞白血病ウィルス遺伝子の検出方法
WO1994026934A2 (fr) * 1993-05-06 1994-11-24 Baxter Diagnostics Inc. Methode de detection du papillomavirus chez l'homme
JPH0775599A (ja) * 1993-09-07 1995-03-20 Japan Synthetic Rubber Co Ltd 標的核酸の検出方法およびそのためのキット

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009034842A1 (fr) * 2007-09-11 2009-03-19 Kaneka Corporation Procédé de détection d'acide nucléique, et coffret de détection d'acide nucléique
JPWO2009034842A1 (ja) * 2007-09-11 2010-12-24 株式会社カネカ 核酸検出方法、および核酸検出キット
US9063130B2 (en) 2007-09-11 2015-06-23 Kaneka Corporation Nucleic acid detection method and nucleic acid detection kit
WO2009054510A1 (fr) 2007-10-25 2009-04-30 Riken Procédé d'amplification isotherme et adn polymérase utilisée dans ce procédé
WO2010061922A1 (fr) 2008-11-27 2010-06-03 独立行政法人理化学研究所 Nouvelle protéine muts et procédé d'utilisation de celle-ci pour déterminer des mutations

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